The objective of this proposal is to elucidate how Thiobacillus ferrooxidans tyrosyl-tRNA synthetase specifically recognizes its cognate transfer RNA, a tRNA with a long extra arm, and discriminates against non- cognate transfer RNAs. By making mutant tRNA genes and tyrosyl-tRNA synthetase variants we propose to define the identity of elements in the transfer RNA responsible for the accurate recognition by the enzyme. In addition, in vitro generated mutants of the enzyme should shed light on the amino acid residues important for binding the substrates ATP, tyrosine, and transfer RNA. The expression of the T. ferrooxidans gene encoding tyrosyl-tRNA synthetase and the search for a second gene for this enzyme will also be undertaken. These studies will complement and extend our ongoing project on tRNA recognition centering on the E. coli glutaminyl-tRNA synthetase system.